Method Description

Complement, Total:

An automated method is performed using liposomes as the target for the serum complement system. The dinitrophenyl (DNP)-labeled liposomes are sensitized with antibody to DNP. Serum complement causes lysis and release of entrapped glucose-6-phosphate dehydrogenase. Glucose-6-phosphate dehydrogenase reacts with glucose-6-phosphate and nicotinamide adenine dinucleotide (NAD[+]). NAD(+) is reduced to NADH and the conversion is measured at 340 nm. The assay correlates with the CH50 assay based on sheep red blood cell lysis, has lower variability, and is simpler to perform.(Package insert: Fujifilm Autokit CH50. Fujifilm Wako Pure Chemical Corporation; 04/01/2018; Yamamoto S, Kubotsu K, Kida M, et al. Automated homogeneous liposome-based assay system for total complement activity. Clin Chem. 1995;41:586-590)

Alternative Complement, Pathway Functional:

The Wieslab enzyme-linked immunosorbent assay complement assay for the alternative pathway combines principles of the hemolytic assay for complement activation with the use of labeled antibodies specific for neoantigens produced as a result of complement activation. The micro titer plate strips are coated with lipopolysaccharide. Patient serum is diluted in diluent containing specific blocker to ensure that only the alternative pathway is activated. During the first incubation, the diluted patient serum in the wells is activated by the coating. The wells are then washed and C5b-9 (membrane attack complex: MAC) is detected with a specific alkaline phosphatase labeled antibody to the neoantigen expressed during MAC formation. After a final wash, an alkaline phosphatase substrate is added. The amount of alternative pathway complement activity correlates with the color intensity of the solution and is measured in terms of absorbance (optical density).(Frazer-Abel A, Sepiashvili L, Mbughuni MM, Willrich MA. Overview of laboratory testing and clinical presentations of complement deficiencies and dysregulation. Adv Clin Chem. 2016;77:1-75. doi:10.1016/bs.acc.2016.06.001)

Complements C3 and C4; Factor B and Factor H Complement Antigens:

In these Siemens Nephelometer II methods, the light scattered onto the antigen-antibody complexes is measured. The intensity of the measured scattered light is proportional to the amount of antigen-antibody complexes in the sample under certain conditions. If the antibody volume is kept constant, the signal behaves proportionally to the antigen volume.

A reference curve is generated by a standard with a known antigen content on which the scattered light signals of the samples can be evaluated and calculated as an antigen concentration. Antigen-antibody complexes are formed when a sample containing antigen and the corresponding antiserum are put into a cuvette. A light beam is generated with a light emitting diode, which is transmitted through the cuvette. The light is scattered onto the immuno-complexes that are present. Antigen and antibody are mixed in the initial measurement, but no complex is formed yet. An antigen-antibody complex is formed in the final measurement.

The result is calculated by subtracting value of the final measurement from the initial measurement. The distribution of intensity of the scattered light depends on the ratio of the particle size of the antigen-antibody complexes to the radiated wavelength.(Instruction manual: Siemens Nephelometer II Operations. Siemens, Inc; Version 2.3, 2008; Addendum to the Instruction Manual 2.3, 08/2017)

C4d Complement Activation Fragment:

Microtiter plates are coated with monoclonal antibody specific to the C4d fragment of the fourth component of the complement cascade. Controls, standards, and patient samples are exposed to the plate. After washing the plate, a horseradish peroxidase-conjugated polyclonal C4d antibody is added followed by a substrate to initiate color change.(Package insert: MicroVue C4d Fragment EIA Kit. Quidel Corporation; A009000EN00 10/2017)

CBb Complement Activation Fragment:

Microtiter plates are coated with monoclonal antibody specific to the complement factor Bb (CBb) fragment of the fourth component of the complement cascade. Controls, standards, and patient samples are exposed to the plate. After washing the plate, a horseradish peroxidase-conjugated polyclonal CBb antibody is added followed by a substrate to initiate color change.(Package insert: MicroVue CBb Plus EIA Kit. Quidel Corporation; 027002EN00 09/2016)

SC5b-9 Complement Activation Complex:

Microtiter plates are coated with monoclonal antibody specific to the C9 ring of the SC5b-9 complex. Controls, standards, and patient samples are exposed to the plate. After washing the plate, a horseradish peroxidase-conjugated anti-SC5b-9 complex antibody is added followed by a substrate to initiate color change.(Package insert: MicroVue SC5b-9 Plus EIA Kit. Quidel Corporation; 020001EN00 05/2017)